The use of sensors in the medical field for testing various blood analytes and in the environmental field for monitoring water or soil contamination is well known. Many of these sensors perform an electrochemical measurement by applying a potential difference across two or more electrodes which are in contact with a reagent and sample. Two-electrode sensors are known which include a working electrode and either a counter or a reference/counter ("reference") electrode. Three-electrode sensors are also known which have a working electrode, a counter electrode, and a reference electrode. Since the area of the working electrode in any of the above sensor designs has a direct effect on the amount of current measured, it is highly desirable to fabricate sensors which have a precisely-defined working electrode area.
Fabricating electrodes for use in sensors has been accomplished by cutting and sealing, "thick-film" or "screen printing", and "thin-film" deposition methods (commonly used in the production of integrated circuits). Recently, photolithography has also been used to pattern electrodes on the surface of a substrate. While some of these techniques permit precise electrode sizing and placement on the support substrate, the ability of sensors made from such electrodes to make precise measurements is limited by the definition of the working electrode area.
Printed circuit boards ("PCBs") and flex circuits are widely used in the electronics industry as a means of interconnecting electrical components. There are two basic systems used to produce PCBs and flex circuits. One is called the "additive method" and the other is called the "subtractive method". With the additive method, the desired circuit pattern is built on top of a non-conductive plastic, ceramic, or other substrate. In the subtractive method, a non-conductive substrate (e.g., epoxy bonded fiberglass in the case of PCBs, polyimide in the case of flex-circuits) is laminated with a copper foil. The copper is then patterned using standard photolithography and wet chemical etching techniques. The copper circuit may subsequently be plated with nickel, gold, or other metal.
The metal patterning techniques described above which are common to the PCB industry, however, are unsuitable for biological applications (e.g., analyte sensing). The plating of metal onto a copper-clad substrate, as described above, results in an irregular, granular surface that allows penetration of a biological fluid to the underlying copper, thus giving rise to background electrochemical signals that interfere with measurements. In addition, copper and nickel are themselves electroactive at the potentials commonly used for sensing, and therefore cannot be used as a working electrode.